The prognostic value of vascular endothelial growth factor in 574 node-negative breast cancer patients who did not receive adjuvant systemic therapy.

The growth and metastasising capacity of solid tumours are dependent on angiogenesis. Vascular endothelial growth factor is a mediator of angiogenesis. In this study we investigated whether vascular endothelial growth factor is associated with the natural course of the disease in primary invasive breast cancer. In 574 tumours of patients with node-negative invasive breast cancer the cytosolic levels of vascular endothelial growth factor were measured using a quantitative enzyme-linked immunosorbent assay. These patients did not receive adjuvant systemic therapy and were followed for a median follow-up time of 61 months (range 2-155 months) after the primary diagnosis. Correlations with well-known prognostic factors, and univariate and multivariate survival analyses were performed. Vascular endothelial growth factor level was positively associated with age and tumour size (P=0.042 and P=0.029, respectively). In addition, vascular endothelial growth factor level was inversely, but weakly correlated with progesterone receptor levels (PgR) (r(s)=-0.090, P=0.035). A high vascular endothelial growth factor level (equal or above the median level of 0.53 ng mg(-1) protein) predicted a reduced relapse-free survival and overall survival in the univariate survival rate analysis (for both P=0.005). In the multivariate analysis as well, vascular endothelial growth factor showed to be an independent predictor of poor relapse-free survival and overall survival (P=0.045 and P=0.029, respectively), in addition to age, tumour size and PgR. The results show that cytosolic levels of vascular endothelial growth factor in tumour tissue samples are independently indicative of prognosis for patients with node-negative breast cancer who were not treated with adjuvant systemic therapy. This implies that vascular endothelial growth factor is related with the natural course of breast cancer progression.

EORTC Receptor and Biomarker Study Group Report: a sandwich enzyme-linked immunosorbent assay for vascular endothelial growth factor in blood and tumor tissue extracts.

A four-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) for application in blood (serum and plasma) and tumor tissue extracts was set up within the framework of the EORTC Receptor and Biomarker Study Group (RBSG). Polyclonal antibodies against VEGF165 were raised in chickens and rabbits, and used in a previously described assay format. The assay was validated and characterized for use in serum, plasma and tumor tissue extracts. The resulting VEGF ELISA was found to be specific for VEGF165 and VEGF121, the main isoforms of VEGF. The assay showed good precision and parallelism in serial dilutions of samples. The assay was not susceptible to interference by heterophilic antibodies because avian antibodies (duck anti-chicken and chicken anti-VEGF) were used in the pre-analyte stage and mammalian antibodies (rabbit anti-VEGF and goat anti-rabbit) in the post-analyte stage. In conclusion, a sensitive, robust and specific VEGF ELISA has been developed. Research into the prognostic value of VEGF employing this assay is currently underway.

Twenty years of experience with the steroid receptor external quality assessment program - the paradigm for tumour biomarker EQA studies. On behalf of the EROTC Receptor and Biomarker Study Group.

Estrogen receptor (ER) assays have clinical relevance in selecting women who would benefit from endocrine intervention. As the degree of benefit from endocrine therapy is directly related to the quantity of receptor present in the tumour, the quality of the steroid receptor assays is important. Moreover, since patients entered in multi-centre trials often include stratification based on the receptor status, receptor assays should be comparable between different institutes. ER- and progesterone receptor (PgR)-assays have been evaluated in quality assessment studies for almost 20 years by the EORTC Receptor and Biomarker Study Group. This study analyses our findings over these years and concludes with a recommended minimum structure on which QA schemes for any biomarker could be based.

EORTC external quality assurance program for ER and PgR measurements: trial 1998/1999. European Organisation for Research and Treatment of Cancer.

Steroid receptor assays have clinical relevance in selecting women who would benefit from endocrine intervention. As the degree of benefit from endocrine therapy is directly related to the quantity of receptor present in the tumour, the quality of the steroid receptor assays is important. Moreover, since patients entered in multi-centre trials often include stratification based on the receptor status, receptor assays should be comparable between different institutes. ER- and PgR-assays have been evaluated in quality assessment studies for almost 20 years by the EORTC Receptor and Biomarker Study Group. During the QA trial 1998/1999 results were reported by 42 participants performing the Ligand Binding Assay (LBA) and by 39 participants using the Enzyme Immuno-Assay (EIA) kit. Each participant received a set of 12 QA vials to be analysed two at a time at two-monthly intervals. The between-laboratory CVs of ER LBA and EIA amounted to 40-50%. For PgR the between-lab CVs for the EIA method are lower as compared with LBA but still are approx. 30%. Notwithstanding the high deviation in reported values and the high between-lab CVs, the consistency of the participants over the year is acceptable, which pave the way for calibration. Indeed, after normalization of assay results the mean between-lab CVs dropped to 11% for ER LBA, 14% for ER EIA, 9% for PgR LBA and 11% for PgR EIA. Such a reduction of between-laboratory CVs is an essential requirement for the use of steroid receptor data in multicentre clinical studies.

External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts.

High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 4 degrees C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable between-laboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop.

The urokinase-type plasminogen activator (uPA) is considered to play a key role in the process of invasion and metastasis. In several independent studies, in a variety of cancer types (e.g. of the breast, colon, stomach, lung, ovary), high antigen levels of uPA in tumour extracts have been associated with rapid disease progression. In these studies, different sets of antibodies and standards (often as commercially available uPA ELISA kits) have been used. The standards provided with the different uPA ELISA kits are different from each other in both composition and source. In addition, the different uPA ELISA kits use antibodies which differ in specificity and affinity for the various forms of uPA including pro-uPA, HMW-uPA, LMW-uPA, the aminoterminal fragment (ATF) and complexes with inhibitors (PAI-1 and PAI-2) and the receptor (uPAR). Further, the composition of tumour tissue extraction buffers differ significantly among the published studies. Thus, it is not surprising that the ranges of cytosolic uPA levels reported differ considerably even when measured within the same tumour type. These discrepancies led the EORTC Receptor and Biomarker Study Group, in conjunction with the BIOMED-1 consortium on 'Clinical Relevance of Proteases in Tumour Invasion and Metastasis', to organise a workshop to study the characteristics associated with six different uPA immunoassays (ELISA) used in clinical studies reported in the literature. Although the absolute uPA antigen values measured with the respective uPA ELISA kits differed, high correlations were obtained for any two of the four uPA ELISA kits finally applied to sets of breast cancer cytosol preparations. The preparations used at present as standards in the various uPA ELISA kits are not representative of actual human breast cancer cytosols. Thus absolute standardisation is only possible by using a common reference sample (breast cancer cytosol) and similarly composed ELISA uPA kits. Then it will be possible to generate comparable data on clinical tissue as well as to check for batch-to-batch variations within particular ELISA kits.